: Worldwide, women bear the brunt of the HIV epidemic, and an effective means to prevent HIV transmission to women remains an elusive goal. Achieving this goal is hampered by our incomplete understanding of the early events in the female reproductive tract (FRT) during sexual transmission. In particular, how resident HIV- susceptible and non-susceptible cells affect viral replication, and sense and respond to HIV infection, are not well understood. Although the upper FRT harbors HIV-susceptible cells, is exposed to semen components, and is infected early in SIV transmission models, it is understudied as a portal of entry for HIV. In cycling women, the endometrium of the upper FRT is extensively remodeled in response to the sex steroids estrogen and progesterone. Progesterone and progestins, synthetic mimics of progesterone, are associated with increased susceptibility to HIV infection in humans and in non-human primate models. Therefore, understanding how these compounds affect the endometrium may yield insights into how HIV establishes infection through this tissue. In our preliminary studies, progesterone decreased barrier function of endometrial epithelial cells, which can facilitate entry of HIV from the luminal cavity into the stromal compartment of the endometrium. Within the stroma resides a dense population of endometrial stromal fibroblasts. Although these cells were not permissive to HIV infection, in co-culture with CD4+ T cells they increased infection by up to 81- fold. In contrast, when progesterone was absent, epithelial barrier integrity was maintained, and the intact layer of endometrial epithelial cells restricted infection by eliciting an antiviral state in co-cultured CD4+ T cells. Using an ex vivo system reconstituted from cells from endometrial biopsies, we will examine the molecular mechanisms responsible for progesterone-induced leakiness (Specific Aim 1), the increased susceptibility to infection mediated by stromal fibroblasts (Specific Aim 2), and epithelia-induced viral restriction (Specific Aim 3) in the upper FRT. Because progesterone-induced epithelial permeability may also occur in the lower FRT, we will additionally test this model using cells isolated from ectocervical biopsies. The proposed studies will deepen our understanding of HIV transmission and restriction in the FRT and provide a robust ex vivo model system to examine the complex interplay between sex steroids and tissue-resident cells in the context of HIV susceptibility. This system and the phenotyping tools we have established can also be adapted to study HIV transmission and restriction in other mucosal portals of entry such the intestinal mucosa and the male foreskin.